The major objective of our research is to understand how proteins respond to metabolites and regulate transcription. Our work has focused on the lactose operon. For many years the lac operon of E. coli has served as the paradigm for gene regulation. The lac operon provides one of the best models for understanding how a set of structural genes may be switched on or off depending upon the concentration of metabolites. The key regulatory component of the operon is the lac repressor. The lac repressor, in the absence of extracellular lactose, binds tightly to the operator DNA and prevents transcription of the structural genes required for lactose to be used as a biological fuel. When lactose is present in the medium, the repressor dissociates from the operator allowing transcription of the structural genes. The conformational transitions of the repressor in response to bound ligands provided the basis of allostery. In the previous grant period, we determined the three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer 1-isopropyl-beta-D-thiogalactoside (IPTG) and the lac structures provide a detailed structural model for repressor in the induced and the repressed states and the molecular basis of gene regulation. In addition, the structure of the repressor provides a framework for understanding a wealth of biochemical and genetic information. The overall goal of the current proposal is to build on the biochemical, genetic, and structural data to obtain a more detailed understanding of the specificity of repressor binding and the structural basis for the allosteric response. Using biochemical and structural methods we will explore the structural basis of repressor function. The specific aims of this proposal are to: elucidate the interactions between the lac repressor and its operator and determine if there are differences in the way repressor binds to different operator sites; to understand how mutant repressors can bind to operator with increases affinity; to determine the structural and physical basis for nonspecific binding to DNA; and to examine the structural basis of allosteric signaling by effector molecules as well as mutant repressors that disrupt the signaling. Of all transcriptional regulators the lac system has been the most thoroughly studied. As a consequence the repressor and its complexes with operator DNA and effector molecules have both contemporary and historical importance for the understanding of gene regulation.